credo project newsletter

8/2/2019 CREDO Project Newsletter

http://slidepdf.com/reader/full/credo-project-newsletter 1/20Issues 7 and 8 www.credocluster.info November 2006

COMPRENDO: We got the ballrolling – can we call it quits?The potential for endocrine active chemicals (EACs) tocause serious health problems in humans and wildlifeand, in some cases at extremely low doses, raises thevery real question as to whether their presence in theenvironment allows normal development, reproduc-tion and aging of individuals. The costs of future med-ical treatment of endocrine diseases and disordersthat may be caused by exposure to EACs and thecosts of removal of these compounds from the rele-vant environmental media are likely to be very high.

Three and a half years of COMParative REsearch intothe effects of ENDOcrine disrupters has been com-pleted. The following aims to provide a brief overviewon the project’s achievements, as well as to highlightthe gaps in knowledge in endocrine disruptionresearch and where we tried to fill in the blanks.

COMPRENDO was set up to improve the understand-ing of the impacts of endocrine active compounds onaquatic wildlife and humans. The project’s focus wason the investigation of potential effects of androgenicand anti-androgenic compounds (AACs) in a range ofdiverse test systems covering invertebrate, vertebrateand human-relevant test models. A wide range oforganisms were exposed to EACs with similar testdesigns for a comparative assessment and evaluationon the sensitivity of the model systems and endpointsunder investigation. Doses of the test substancesadministered included environmentally relevant con-centrations. Sediments originating from field-samplingin European regions with low and high environmentalquality standards (Falandysz et al. 2006) were used inmixture studies to assess the responses of amphib-ians, crustaceans and molluscan test species for com-parable patterns in biological effects induced by toxicand hormonally active chemicals. Baseline endocrinol-ogy research was a key activity and was conductedto address gaps in knowledge and to advance our

Cluster of research into endocrine disruption in Europe

Coordinating European Environmental and Human Health Research into Endocrine Disruption

n ew s e er Continued on page 2

The end of CREDOWith many of the participating projects completed, theCREDO cluster is now nearing the end of its lifetime.For this, the last issue of the newsletter, we haveasked partner projects for a summary of their findings,and the response has been overwhelming. We havereceived enough material to fill more than a doubleissue. Without a doubt, the cluster has greatly con-tributed to improving our knowledge about endocrinedisruption. This could not have been possible withoutthe enthusiasm of all the labs and workgroupsinvolved and their willingness to collaborate acrossproject boundaries. The last years have shown that wehave succeeded in creating a research focus forendocrine disruption in Europe and beyond.

This is also the time to look ahead to the future. Thefounder projects of the cluster were set up to providean integrated approach to wildlife and human healthproblems, and the cluster has shown how powerfulsuch an approach can be. The question now in manypeoples’ minds is whether we will have the opportu-nity to build on this expertise in the forthcomingSeventh Framework Programme.

I would like to thank the cluster for all the support andgoodwill during these four years.

Andreas Kortenkamp Cluster coordinator

In this edition

COMPRENDO: we got the ball rolling

EURISKED: multi organic risk assessment

UV filters target thyroid biosynthesis

News from EDEN

Bone development and homeostasis

What have we learned from ENDOMET?

EASYRING: non-invasive testing methods

EDERA: more predictive risk assessment

Genetic markers in testis endocrine disruption

Biomimetic and biological sensor systems

Environment and health research under FP7

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COMPRENDO

understanding of the possibilities for applying new animalmodels to predict for possible human health effects. This workalso set out to investigate new test species for chemical test-ing. Residue analyses of EACs in foodstuffs, for all participat-ing EU countries of the COMPRENDO consortium, was report-ed recently in the July 2006 issue of the CREDO newsletter.

Ladykillers in a man’s world?Sexual differentiation and development are endpoints report-ed to be especially susceptible to alterations from exposure toendocrine disrupting chemicals. COMPRENDO investigatedhow susceptible sexual determination, differentiation anddevelopment were to EACs in a range of different testspecies. Was it possible to produce animals of an indetermi-nate gender (with sexual organs that were neither explicitlymale nor explicitly female) on exposure to AACs? We investi-gated this with more than 30 chronic laboratory-based expo-sure experiments. We carried out numerous assays forenzyme activity and steroid determinations and analyses ofEAC residues in human tissues and cell lines, rats, amphib-ians, fish, echinoderms, crustaceans and molluscs. Theresults of these experiments demonstrated that all test sys-tems were affected for the test compounds TBT, TPT, linuron,diuron, fenarimol, vinclozolin, p,p’-DDE. Androgen mimickingsubstances, such as organotins and some pesticides, result-ed in the induction of virilization, reduced reproductive per-formance, accelerated sexual maturity, reduced larval devel-opment, reduced skeletal density, abnormal sex ratios,changes in sex steroid titres and enzyme activities and vari-ous effects on the reproductive organs under histopathologi-cal investigation. Conversely, feminisation effects, includingreduced egg diameters, and a lack of spermatids and sper-matophores in males were observed under anti-androgenexposure. In most of the experiments, the observed effectconcentrations were in the environmentally relevant range.

COMPRENDO's contribution to the ‘OECD testing toolbox’Our studies suggested that the reproductive and develop-mental effects of the test compounds were often comparablebetween invertebrate groups and with the vertebrates stud-ied. Indeed, the data generated suggested that some of ourinvertebrate test models might provide powerful surrogatesfor vertebrate species for the identification of endocrine haz-ards in Tier 1 testing. Two of the organisms, specifically thecalanoid copepods ( Acartia tonsa ) and the prosobranch snailPotamopyrgus antipodarum , used in COMPRENDO are being

considered by the OECD as suitable test organisms for theOECD guideline programme on testing of chemicals (includ-ing for the assessment of endocrine disruption). The draftOECD method on calanoid copepods ( Acartia tonsa ) has beenprepared and a ring-test for validation is currently running(OECD 2005; Kusk & Wollenberger 2005). The prosobranch

snail Potamopyrgus antipodarum has been agreed by theOECD Ad hoc Expert group on Invertebrate Testing as the pre-ferred molluscan species for the development of a test guide-line. As a first step toward validation and generation of a draftguideline with P. antipodarum, COMPRENDO prepared astandard operating procedure (SOP) for the culture of thisspecies, and a SOP for a 56 day reproduction test (water andsediment; Schmitt et al . 2006; the document is available onrequest from the co-ordinator). We await to see which of theEuropean governments will support and drive the implemen-tation of the snail test at the OECD.

Of mice and men…Animal models are used in virtually every field of biomedicalresearch. Almost all medical knowledge and treatment, espe-cially that in the last century, has involved work with laborato-ry animals. While the past 50 years has seen, remarkableadvances in medicine, new models for endocrine diseasesare needed and the COMPRENDO work sought to developsome of these.

The significance and ability to extrapolate results obtainedfrom animal models to human health is an area that needs fargreater research. A good knowledge of comparative anatomy,physiology and function is essential for the animal model ofinterest. Within COMPRENDO we gathered extensive datasets of this nature for several test species. Animal modelsmay be found throughout the animal kingdom, and knowl-edge about human physiology has been achieved in speciesthat seem far removed from the human in terms of evolu-tionary development. A good example is the importance ofthe fruit fly for the original studies of basic genetics.

The EU chemical strategy (REACH) proposes that industrialchemicals are managed more effectively in order to protecthuman health and the environment. Our results support theneed for EU legislation to drive the precautionary principle forsubstances with endocrine-disrupting properties. Currentlythe number of vertebrate animal experiments proposedunder REACH is unacceptably high, emphasising further theneed for suitable non-vertebrate tests (as developed underCOMPRENDO).

Comparative EndocrinologyCOMPRENDO has also made efforts to elucidate the similar-ities and differences in steroid metabolism in a wide range oftest species and animal phyla (Janer et al 2005a-d, Janer et al.

2006, Lavado et al. 2006). It is important to note that as wellas the striking similarities between vertebrate and inverte-brate responses to AACs, there are also differences in thepatterns of responses to the different chemicals. For examplethe effects of AACs on steroidogenesis differ between fishand molluscs and between molluscs and crustaceans.

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COMPRENDO

COMPRENDO has brought two new invertebrate species tothe OECD for consideration and has developed a multi-fishspecies gene array for the enhancement of chemical testingregimes and systems. These tools are also likely to improvethe protection of human health, for example as applied to themonitoring of water quality and effectiveness of wastewatertreatment plants, or in the assessing for compliance andthreshold of biological effects of chemicals and their mixtures.

The work under COMPRENDO has further served to highlightthat we perhaps need to be less restrictive in our approach tochemical testing. Understanding the basic biology andendocrinology of a greater number of animals (both inverte-brate and vertebrates) is likely to help us to develop moreeffective and sensitive testing systems for chemicals of con-cern, such as EACs, and thus develop more effective warningsystems for the adverse health effects of chemicals. It shouldalso be emphasised that an understanding of chemical effectsin invertebrates is not important only for their potential useand development as surrogates for some aspects of verte-brate testing, but also for the protection of invertebrate faunain the environment per se , as they play a pivotal role in allecosystems. Little is known about potential effects ofendocrine disrupters in wild invertebrate populations andthere is the very real need for field- & bio-monitoring studiesto investigate for possible impacts of EACs on invertebrates,as has been shown for some vertebrate populations.

COMPRENDO was a journey of discovery. The ambitious anddiverse nature of the work under COMPRENDO meant thatre-iterative evaluation of the work, needs, and achievementswas essential throughout the life of the project. Delivery onmany aspects of the science was made greater through link-ages of the work with other research projects and indeednecessitated some resourcefulness on behalf of the partners.In the rapidly moving science of molecular biology and genearrays, team members also had to be highly adaptive and flex-ible to deliver on some of the challenges set. COMPRENDOwas European research at its best. In addition to a valuablecontribution to our understanding of endocrine disruption,COMPRENDO has also served to forge strong internationalresearch linkages between member states of the EU and todevelop great friendships. To those who funded and support-ed COMPRENDO, we should like to express our sincere grat-itude. We hope the European Union, as our benefactor, willhave the staying power that will inevitably be required toutilise the findings from COMPRENDO and from other proj-ects in the CREDO cluster to more forward-thinking politicalactions to regulate hormone mimicking substances.

Ulrike Schulte-Oehlmann, Susan Jobling, Charles Tyler andJörg Oehlmann on behalf of the COMPRENDO consortium

www.comprendo-project.org

References

Crofton, K.M., Craft, E.S., Hedge, J.S., Gennings, C., Simmons, J.E.,Carchman, R.A., Carter Jr., W.A., DeVito, M.J. (2005). Thyroid-hormone-dis-rupting chemicals: Evidence for dose-dependent additivity or synergism.Environ. Health Perspect. 113: 1549-54.Falandysz J., Albanis T., Bachmann J., Bettinetti R., Bochentin I., Boti V.,Bristeau S., Daehne B., Dagnac T., Galassi S., Jeannot R., Oehlmann J.,Orlikowska A., Sakkas V., Valsamaki V. & Schulte-Oehlmann U. (2006).Some chemical contaminant of surface sediments at the Baltic Sea coastalregion of the Gulf of Gda´ nsk, the Brda River outlet in inland Poland, and at ref-erence sites in the Wadden Sea, Germany, with special emphasis on andro-genic and anti-androgenic compounds. J. Environ. Sci. Health: submitted.

Filby, A.L., and Tyler, C.R. (2005). Molecular characterization of estrogenreceptors 1, 2a and 2b and their tissue and ontogenic expression profiles in fat-head minnow (Pimephales promelas). Biol. of Reprod. 73:648-662.

Filby, A.L., Thorpe, K.L. and Tyler, C.R. (2006). Multiple molecular effect path-ways for an environmental oestrogen in fish. J. Mol.Endocrinol.Accepted, in press.

Fisher J.S. (2004). Environmental anti-androgens and male reproductivehealth: Focus on phthalates and testicular dysgenesis syndrome. Reproduction127: 305-15.

Harris, C.A., Santos, E.M., Janbakhsh, A., Pottinger, T.G., Tyler, C.R.,Sumpter, J.P. (2001). Nonylphenol affects gonadotropin levels in the pituitarygland and plasma of female rainbow trout: Environ. Sci. Technol. 35: 2909-2916.

Janer, G., LeBlanc, G.A. & Porte, C. (2005a). Androgen metabolism in inver-tebrates and its modulation by xenoandrogens. A comparative study. Ann. N.Y.Acad. Sci. 1040: 354-356.

Janer, G., LeBlanc, G.A. & Porte, C. (2005b). A comparative study on andro-gen metabolism in three invertebrate species. Gen. Comp. Endocrinol. 143:211-221.

Janer, G., Lavado, R., Thibaut, R. & Porte C. (2005c). Effects of 17b-estradi-ol exposure in the mussel Mytilus galloprovincialis: A possible regulating rolefor steroid acyltransferases. Aquat. Toxicol. 75: 32-42.

Janer, G., Sternberg, R.M., LeBlanc, G.A. & Porte, C. (2005d). Testosteroneconjugating activities in invertebrates: Are they targets for endocrine disrup-tors? Aquat. Toxicol. 71: 273-282.

Janer, G., Bachmann, J., Oehlmann, J., Schulte-Oehlmann, U. & Porte, C.(2006). The effect of organotin compounds on gender specific androstene-dione metabolism in the freshwater ramshorn snail Marisa cornuarietis. J.Steroid. Biochem. Mol. Biol. 99: 147-156.

Janer, G., Lyssimachou, A., Bachmann, J., Oehlmann, J., Schulte-

Oehlmann, U. & Porte, C. (2006). Sexual dimorphism in esterified steroidlevels in the gastropod Marisa cornuarietis: The effect of xenoandrogeniccompounds. Steroids, 71: 435-444.

Kusk, K.O., Wollenberger, L. (2005). Validation of a full life-cycle test with thecopepod Acartia tonsa. Institute of Environment & Resources, TechnicalUniversity of Denmark, 56 pp.

Lavado, R. Barbaglio, A., Candia Carnevali, M.D. & Porte, C. (2006). Steroidlevels in crinoid echinoderms are altered by exposure to model endocrinedisruptors. Steroids, in press.

Moggs, J.G., Tinwell, H., Spurway, T., Chang, H-S., Pate, I., Lim,F.L., Moore,D.J.,Soames, A., Stuckey, R., Currie, R., Zhu, T., Kimber, I.,Ashby,J., Orphanides, G. (2001). Phenotypic anchoring of gene expression changesduring estrogen-induceduterine growth.Environ. Health Perspect. 112: 1589-1606.

Perou, C.M., Serlie, T., Eisen, M.B. (2000). Molecular portraits of humanbreast tumours. Nature 406 (6797): 747-752.

Schaner, M.E., Douglas, T.R., Ciaravino , G., Sørlie, T., Troyanskaya,O.,

Diehn, M., Wang Y.C., Duran, G.E., Sikic , T.L., Caldeira, S., Skomedal, H.,Tu, I-P., Hernandez-Boussard , T., Johnson, S.W., O'Dwyer, P.J., Fero, M.J.,Kristensen, G.B., Børresen-Dale, A.-L., Hastie, T., Tibshirani, R., van deRijn, R., Teng, N.N., Longacre, T.A., Botstein, D., O. Brown, P., Sikic, B.I.(2003). Gene expression patterns in ovarian carcinomas. Environ. HealthPerspect. 112:1589-606.

OECD (2005). OECD Draft Guidelines for Testing of Chemicals. Proposal for anew Guideline. Calanoid Copepod Development and Reproduction test withAcartia tonsa. 2005. Paris, France, Organisation for Economic Cooperation andDevelopment. Edited by Kusk, K.O. and L. Wollenberger.

Opitz, R., Braunbeck, T., Bögi, C., Pickford, D.B., Nentwig, G., Oehlmann,J., Tooi, O., Lutz, I. & Kloas, W. (2005). Description and initial evaluation of aXenopus metamorphosis assay for detection of thyroid system-disruptingactivities of environmental compounds. Environ. Toxicol. Chem. 24: 653-664.

Schmitt, C., Duft, M., Brandelik, C., Schulte-Oehlmann, U., Oehlmann, J.(2006). SOP for testing of chemicals: Reproduction test with the prosobranch snailPotamopyrgus antipodarum for testing endocrine active chemicals. Part I: Culturing

of Potamopyrgus antipodarum. Part II: Reproduction test using water exposure.Part III: Reproduction test using spiked sediment. J.W. Goethe University ofFrankfurt, Department of Ecology and Evolution – Ecotoxicology, Germany.

Schulte-Oehlmann, U, Watermann, B., Tillmann, M., Scherf, S., Markert,B., Oehlmann, J. (2000). Effects of endocrine disruptors on prosobranch snails(Mollusca: Gastropoda ) in the laboratory. Part II: Triphenyltin as a xeno-andro-gen. Ecotoxicology 9: 399-412.


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EURISKED

Multi-organic risk assessment of selected endocrine disrupters

Objectives

The ultimate goal of the EURISKED project was to determinemulti-organic effects of a variety of endocrine disrupters(EDs). These are: nonylphenol, bisphenol A, dibutylphtalate,octyl-methoxycinnamate (OMC), 4-methylbenzylidene cam-phor (4-MBC), benzophenone-2, procymidon, linuron, resver-atrol, 8-prenylnaringenin, genistein, oestradiol-benzoate, andandrostandiol.

Utilizing a variety of reporter cell systems, the first goal ofthe project was to determine steroidal (oestrogenic, andro-genic, thyromimetic gluco- or mineralocorticoid or progesta-

tional) effects. On the basis of these results, reporter genetransfected mice were to be investigated with those sub-stances which proved to have effects in the cell biologicalexperiments. In parallel to these reporter cell assays, experi-ments in immature and adult female and male rats as well asin steroid receptor deleted (K.O.) animals were performed.Effects of the endocrine disrupters were to be measured byphysiological, molecular and morphological means. Steroidsand also thyroid hormones have profound effects in manyother organs outside the reproductive tract. It was thereforethe major goal of this project to study these effects outsidethe reproductive tract. As a control, targets within the repro-ductive tract needed to be incorporated in the study design

as well.

Scientific achievements

There is now clear indication that a number of the test sub-stances, which are widely used as food additives, cosmeticsor plasticizers have profound effects on many organs. Thiscould be verified in the various reporter assays utilizing eithergene transfected cells from different organ-specific primarycells with specific responses to steroids, or reporter genetransfected mice. The experiments with the reporter cellswere completed within the first two years, those with thereporter animals were finished by the end of the project.

Utilizing these assays, it became clear that the endocrine dis-rupters had effects in a variety of cells stemming from dif-ferent organs. Some are oestrogenic, some anti-androgenic,others are unexplainable at present and may involve othernuclear or non-nuclear receptor mediated processes.

As expected, many of the proposed EDs have effects out-side of the reproductive tract. So far, the brain, the pituitarygland, the liver, the bone, fat tissue and – of major impor-tance – the thyroid gland were shown to be target of someof the studied EDs. Plant derived so-called phytoestrogensstimulate bone formation but also uterine and mammarygland tissue. By means of histology, including immunocyto-

chemistry, it was shown that all uterine parts (endometrium,myometrium) were stimulated by the phytoestrogens,whereas 4-MBC and OMC had very mild uterotropic effects.Linuron and procymidone were ineffec tive. 8-Prenylnaringenin, a hop-derived flavonoid, caused formation

of polypoid structures in the endometrium, an effect whichhas never been published before.

In the mammary gland all phytoestrogens clearly stimulatedthe formation of the the nuclear proliferation markerProliferating Cell Nuclear Antigen (PCNA) and of the proges-terone receptors, both are very typical oestrogenic effects.The higher doses of the phytoestrogens stimulated alsodevelopment of mammary gland ducts and milk production.If occurring also in the human this would endanger theuterus and the mammary gland to develop malignanttumours.

In the thyroid, the phytoestrogens and the UV screens inhib-ited either thyroidperoxidase or the iodide symporter, and inother organs the thyroid hormone deiodinases were affect-ed, which may endanger the organism to become hypothy-roid. Some of the tested UV filters proved to be oestrogenic,an undesired effect if the substances are resorbed transcu-taneously. Upon approval through the ethical committee, fivefemale and six male human probands were subjected totreatment with two commercially available sunscreens witha high UV protection factor. One company responded to therequest about information on the amounts of OMC in theirproduct. Surprisingly 10% of the whole sun milk was OMC.The other sunscreen contained OMC and 4MBC in unknown

amounts. Extraction of these substances and HPLC separa-tion and UV detection indicated that up to 10g of 4MBC andOMC were present in 100ml of the second commerciallyavailable sunscreen. The back and front part of the trunk ofthe test subjects were exposed to known amounts of the UVscreens and blood samples were collected throughindwelling antecubital vein catheters prior to and up to 8hours after initial exposure. Exposure was then continued foranother 3 days and on the fourth day the same treatmentand blood withdrawal regimen was applied. Extraction, HPLCseparation and UV detection did not detect any of the twosuncreens. In animal experiments it was also shown thatboth sunscreens are rapidly metabolized and also the

metabolite could not be detected in the serum of the subject.Extracts of selected blood samples were subjected to thehighly sensitive method of mass spectrometry and again noindication of presence of either of the two UV screens ortheir metabolites could be demonstrated. Hence, the meas-urement performed so far did not result in detectable serumlevels.

The two pesticides tested inhibited testosterone-inducedgrowth of prostates and seminal vesicles. This anti-andro-genic effect may have adverse effects in pubertal boys, notonly in the development of prostate or seminal vesicles butalso in bone maturation.

Prior to the establisment of the EURISKED project it wasshown that the prostate expresses high amounts of theoestrogen receptor of the beta subtype (ER β). It was there-fore another focus of interest to investigate the effects of



EURISKED

ERβ in the prostate. By means of histological and immuo-cytochemical analysis and utilizing the ER β knock-outmouse model, it could be demonstrated that the ER βappears to have anti-proliferative effects in the prostate,thereby having possibly protecting effects against thedevelopment of prostate cancer. From the two UVscreens, 4MBC proved to be a pure ER β agonist, whichmay therefore have a potential in the treatment of prostatecancer. In a detailed study about antenatal effects of thesunscreens, neuronal and maturational parameters wereinvestigated and 4MBC at a relatively high dose exertedclear effects in the animals which were perinatally treatedand investigated at the age of 12 weeks. Significanteffects were observed in the prostate, thyroid gland,uterus, ovary and also molecular endpoints like IGF1, prog-esterone gene expression were altered. In another exper-iment, the effects of 4MBC and benzopheneone-2 onpubertal development was investigated and a delay ofpuberty and a variety of adverse effects in a number oforgans including the uterus and the bone were observed.

Two consortium members investigated the effects of thearylhydrocarbon receptor (AhR) in some more detail. Itcould be shown that genistein and resveratrol were AhRagonists, while 17 β-oestradiol, 4MBC and OMC were AhRantagonists. The biological meaning of these findings arecurrently unclear. Stimulation of the AhR results in forma-tion of CYP1A1 enzymes which are involved in detoxifiac-tion of environmental toxicants.

Socio-economic relevance and policy implications

The consortium is confident that the results furthered ourknowledge about the safety of some food additives, pesti-cides and of some UV screens, which will lead to politicalactivities to direct the use of these compounds.Information made available to consumers will help themmake balanced decisions about their risks of exposure tothese chemicals.

Conclusions

It appears that the choice of EDs and of animal modelswas appropriate and could lead to recommendationsresulting in risk assessment guidelines for humans. Theconsiderable amount of samples generated during thefunding period of the project as well as scientific publica-tions documenting the results of the project are deliver-ables of the project.

Wolfgang WuttkeDivision of Clinical and Experimental Endocrinology,University of Göttingen, Germany, on behalf of the

EURISKED consortium.

www.eurisked.org

Thyroid hormone biosynthesisA target for endocrine disruption by UV filters

In recent years it has become clear that, in addition to thereproductive system, the thyroid hormone axis is a major tar-get of endocrine disruption. In our investigations performedas part of the EURISKED project we examined the effects ofselected compounds, including certain UV filters, on thyroidhormone biosynthesis. UV filters are produced in amounts ofseveral hundreds of tons per year, and their main use is inskin protection products against sunburn, photo aging andskin cancer. For example, 4-methylbenzylidene-camphor (4-MBC) is contained at concentrations of up to 4% (w/w) incommercial preparations. Another UV filter, benzophenone 2(BP2), is no longer permitted in sun lotions within the EU,however it is still contained in other cosmetics to preventtheir UV-induced damage. The concentrations of BP2 inthese cosmetics are much lower than the concentrationsallowed for other UV filters in sun protection products, butsince BP2 is found in a wide variety of products, it is likelythat it is applied to human skin virtually every day.

In vitro and in vivo effects of 4-MBC and BP2 on thethyroid hormone axis

A prerequisite for the synthesis of the iodide-containing thy-roid hormones is the uptake of iodide into the hormone-pro-ducing thyroid cells. This process is catalyzed by the so-called sodium iodide symporter (NIS) and can be examined ina thyroid cell culture model, the rat-derived cell line FRTL-5.We found that 4-MBC decreased iodide accumulation in thiscell line in a dose-dependent manner, starting at a concen-tration as low as 100µmol/L. This effect was due to areduced expression of the NIS protein, as determined byWestern blotting.

Thyroid hormone biosynthesis is catalyzed by thyroid peroxi-dase (TPO). We developed an in vitro enzyme assay basedon human recombinant TPO (hrTPO) which was stably trans-fected into the human thyroid carcinoma cell line FTC-238.Using this assay we demonstrated that BP2 inhibited hrTPOwith IC

50values of 450 and 370µmol/L, respectively, in the

guaiacol and the iodide oxidation reaction, two classicalmethods to measure peroxidase activities. These IC 50 valuesare one to two orders of magnitude lower than those deter-mined for methimazole (MMI) and propylthiouracil (PTU),two TPO inhibitors used as anti-thyroid drugs in the therapyof hyperthyroidism (figure 1). Preincubation of hrTPO with acombination of BP2 and hydrogen peroxide (a necessarycofactor for TPO), but without substrate, inactivated theenzyme. This effect was prevented by the presence ofmicro-molar amounts of iodide in the preincubaton mixture,indicating that effects of BP2 on TPO may be especially detri-mental under conditions of iodide deficiency.

To examine the effects of the two UV screens in vivo , ratswere treated with increasing doses of 4-MBC and BP2.Already after 5 days application, both compounds dose-dependently decreased T4 and increased TSH serum levels.This is a constellation typical for hypothyroidism, a condition



Consequences

A ban of 4-MBC is already under consideration by EU author-ities and our findings have deepened the concern over thissubstance. As for BP2, data on human exposure and skinabsorption are needed in order to facilitate a new evaluationof the risks of its use as a UV stabilisator in cosmetics.

Cornelia Schmutzler, Inka Hamann and Josef KöhrleInstitute for Experimental Endocrinology, Charité Berlin,Germany

www.eurisked.org

References

Janjua NR, Mogensen B, Andersson AM, Petersen JH, Henriksen M,Skakkebaek NE, Wulf HC (2004). Systemic absorption of the sunscreensbenzophenone-3, octyl-methoxycinnamate, and 3-(4-methyl-benzylidene) cam-phor after whole-body topical application and reproductive hormone levels inhumans. J. Invest. Dermatol. 123, 57-61.

Jarry H, Christoffel J, Rimoldi G, Koch L, Wuttke W (2004). Multi-organicendocrine disrupting activity of the UV screen benzophenone 2 (BP2) inovariectomized adult rats after 5 days treatment. Toxicology 205, 87-93.

Schlumpf M, Schmid P, Durrer S, Conscience M, Maerkel K, Henseler M,Gruetter M, Herzog I, Reolon S, Ceccatelli R, Faass O, Stutz E, Jarry H,Wuttke W, Lichtensteiger W (2004). Endocrine activity and developmentaltoxicity of cosmetic UV filters-an update. Toxicology 205, 113-122.

www.credocluster.info November 2006

of pathologically low thyroid performance. 4-MBC alsocaused goitre in rats, both after short-term (5 days) and long-term (12 months) administration in rats (Jarry et al . 2004;Schlumpf et al . 2004; Inka Hamann, unpublished).

Risks for human health?

Several recent publications, among them Janjua et al. (2004),show that UV filters can penetrate the human skin, and in thecase of 4-MBC, reach serum levels of ~80µmol/L. There areno data for BP2, but benzophenone 3, which is chemicallyclosely related to BP2, was detected in human serum at aconcentration of ~1.3µmol/L. These are concentrations atwhich effects were seen in our in vitro assays. Thus, it can-not be excluded that UV filters may also have adverse effectson thyroid hormone biosynthesis in humans. According toour data, the effects of UV filters on TPO and on the NIS maycombine with and aggravate effects of low iodine supply.This finding has to be considered in the context that iodinedeficiency is still a problem in many parts of the world(including industrialised countries such as Germany).

a b c

Figure 1: Dose response curves and IC 50 values for the inhibition of hrTPO by a) MMI, b) PTU and c) the UV filter BP2 in the guaiacol

oxidation assay.

EURISKED EDEN

The EDEN project has recently been granted an extension ofsix months, to the end of May 2007. This was triggered byforce majeur delays in catching fish and obtaining tissuespecimens. The extension will enable EDEN to completeurgently needed exposure assessments for multiple chemi-cals in fish and human tissues. Such assessments were rarelycarried out in the past. Instead, single chemicals were meas-ured, leaving the issue of combined exposures unanswered.

The development of new biomarkers useful for examiningendocrine disruption in humans is making progress, as arestudies of the role of the KISS peptin in puberty. New dataabout sperm quality in Germany await final analysis, due toappear in 2007.

NEWS FROM EDENConsiderable progress has also been made with our under-standing of how endocrine disrupters work together asmixtures, and current work focuses on the interplay ofsubstances with dissimilar modes of action. An expert work-shop was held to explore options of incorporating this newknowledge into better chemicals regulation. The EDEN teamis currently busy with finalising a document about mixturesregulation which will be published in 2007.

Andreas KortenkampUniversity of LondonSchool of Pharmacy, UK

www.edenresearch.info



Bone quality and function have not traditionally been thefocus of toxicological research. However, in recent years anumber of chemicals and food contaminants have been iden-tified that interfere with bone development and remodelling.Among those it has been demonstrated that aryl hydrocarbonreceptor (AhR) ligands, such as 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), have the ability to alter bone structure andfunction at doses that are close to human background expo-sure levels.

The BoneTox project aims to clarify whether AhR-mediatedendocrine mechanisms can explain the observed bone toxici-ty. Alterations in heart and tooth development are additional

physiological outcomes studied in the project. The molecularfocus of the project is on the dioxin, oestrogen and retinoid(vitamin A) signalling pathways. This article covers bone andretinoid results that have been or are in the process of beingpublished.

Methods and experimental modelsMethod development is essential to the BoneTox projectsince few molecular biology and chemical methods havebeen adapted to the analysis of hard tissues. Similarly, bonemeasurements of small animals are only standardised to alimited extent to accommodate for toxicological studies.

The animal models cover both traditional experimental lines ofrats and mice, and several genetically modified mouse strains.In addition, human epidemiological studies are ongoing, but toa limited extent.

Bone studies in ratsCombined gestational and lactational TCDD exposure hasshown bone defects mainly in the offspring of the most sen-sitive rat line studied (Miettinen et al. , 2005). The defectsincluded decreases in bone mineral density (BMD), bonelength and cross-sectional area of cortex. Mechanical testingrevealed significantly reduced breaking force and stiffness ofthe bones. The effects were time- and dose-dependent, with

earlier exposure resulting in more severe defects. Most of thedefects were almost recovered in one-year old offspring.These results indicate that dioxins affect the developingbones by interfering with bone growth and mechanicalstrength, and that the effects are mainly reversible.

The effect on bone of early life exposure to a chemical mix-ture that mimics pollutants found in the blood of the CanadianInuit population has been studied in the rat (unpublished data).Preliminary results indicate that several bone parametersrevealed from pQCT measurements were affected both indams and offspring at doses of about 500 times human intakelevels. Changes observed in the offspring were evident in

young and young adult pups, whereas older offspring showedclear, although not complete, recovery.

Effects on bone by the brominated flame retardants hexabro-mocyclododecane (Olausson et al. , 2006), brominated

diphenyl ethers (pentaBDE (Litens et al. , 2006) and decaBDE)and tetrabromobisphenol A are studied in a series of 28-daysrepeat dose and/or 1-generation toxicity studies in the rat.This work is done in collaboration with the EC project FIRE(QLRT-2001-00596).

Bone studies in miceSex steroids are important in maintaining the skeleton of both

males and females. BoneTox partners have been characteris-ing the bone phenotypes of oestrogen receptor α and β (ERαand ER β) inactivated mice (Moverare et al. , 2003). These stud-ies demonstrated that ER α is the most important oestrogenreceptor for the regulation of several different bone parame-ters including trabecular thickness, trabecular number andcortical thickness. To obtain a global perspective of the effectof oestrogen on bone proteins, whole bones from control andovariectomised mice treated with oestradiol were used forproteome analysis (Pastorelli et al. , 2005). Several noveloestrogen-regulated proteins were found, and the changes inthe bone protein profile suggest that oestradiol affects abroad spectrum of metabolic pathways in bone. Bone studies

in other mouse lines, modified with regard to RAR, AhR andRALDH1are ongoing.

Bone studies in vitro Analyses of the localization and action of AhR in bone cellshave revealed a strong expression of AhR both in osteoblastsand osteoclasts (Ilvesaro et al. , 2005). The activity of osteo-clasts was not affected by TCDD exposure, indicating thatTCDD does not have direct effect on mature osteoclasts.However, continued studies have shown that TCDD couldeffectively disturb the osteoclastogenesis (unpublished data).

Retinoids

The retinoid (vitamin A) system plays an essential role inembryonic development, reproduction, growth and overallsurvival (Mark et al. , 2006). Scientists in BoneTox have clearlydemonstrated that the retinoid system is a sensitive target forTCDD and other AhR ligands (Fletcher et al. , 2005a; Nilsson

BONETOX

Figure 1. Peripheral quantitative computed tomography (pQCT) scans

on tibial and femoral diaphysis in rats on postnatal day (PND) 35 after different maternal doses (0, 0.1, 0,3, 1 µg/kg) of TCDD displaying the changes in cross-sectional geometry. (Image by Hanna Miettinen)

Bone development and homeostasis – new critical targets in toxicology

Tibia

Femur

Maternal dose of TCDD (µg/kg)



and Hakansson, 2002). Altered tissue levels of multiple

retinoid forms, as well as altered retinoid specific enzymeactivities and expression profiles have been observed at lowAhR ligand exposure levels. Through the collaboration withFIRE new knowledge will be gained about the structuralrequirements for disturbances in the retinoid system bybrominated flame retardants, some of which are suspected tointerfere with the AhR.

A new retinoic acid metabolite, 9-cis-4-oxo-13,14-dihydro-retinoic acid has been identified as a novel and sensitive indi-cator of TCDD exposure (Schmidt et al., 2002, Fletcher et al.,2005a). The liver level of this metabolite was markedlydecreased in TCDD-treated rats at all timepoints analysed in a

112 day time-course single dose study (unpublished data).The metabolite level was also decreased in the liver of damsand offspring exposed to a chemical mixture based on theblood profile seen in the Canadian Inuit population (unpub-lished data). The biological function and activity of the metabo-lite is currently being further investigated.

To identify novel genes and pathways that may be associatedwith dioxin-induced retinoid disruption, global analyses ofgene expression after exposure to TCDD has been carried out(Fletcher et al. , 2005b), and studies have been performed inwildtype and mutant mice lacking one or more isotypes ofRAR, RXR or intracellular binding proteins for retinoids

(CRABP1, CRABP2, CRBP1). Treatment with TCDD resultedin loss of approximately the same amount of retinoids inthe different receptor knockout lines, with the exceptionof RXRβ-null mice, which did not lose any retinoids uponTCDD exposure (Hoegberg et al., 2005). Mice lacking all three

intracellular binding proteins for retinoids displayed signifi-cantly lower hepatic retinoid levels compared to wild-typemice, and following TCDD exposure these mice were almostentirely depleted for hepatic retinoid stores (Hoegberg et al.,2005).

DisseminationAll the results gained in the BoneTox project are disseminat-ed during scientific meetings and are published promptly inscientific journals. Several thesis projects have been conclud-ed while others are still ongoing. A special session on boneand tooth toxicology, with many BoneTox contributors, hasbeen held at the Dioxin 2006 conference in Oslo, Norway. TheBoneTox project will be finalized at the end of 2006.

Louise Lyrenäs, Maria Herlin and Helen HåkanssonInstitute of Environmental Medicine, Karolinska Institutet,Sweden, on behalf of all BoneTox participants.

www.imm.ki.se/bonetox/

References

Fletcher N, Giese N, Schmidt C, Stern N, Lind PM, Viluksela M, TuomistoJT, Tuomisto J, Nau H, Hakansson H. (2005a). Toxicol Sci. 86(2), 264-72.

Fletcher, N., Wahlstrom, D., Lundberg, R., Nilsson, C.B., Nilsson, K.C.,Stockling, K., Hellmold, H. and Hakansson, H. (2005b). Toxicol ApplPharmacol 207, 1-24.

Hoegberg, P., Schmidt, C.K., Fletcher, N., Nilsson C.B., Trossvik, C.,Schuur, A.G., Brouwer, A., Nau, H., Ghysenlinck, N.B., Chambon, P.,Håkansson, H. (2005). Chem Biol Interact 156, 25-39.

Ilvesaro, J., Pohjanvirta, R., Tuomisto, J., Viluksela, M. and Tuukkanen, J.(2005). Life Sci 77, 1351-66.

Litens, S., Lyrenäs, L., van der Ven, L. and Håkansson, H. (2006). in: Dioxin2006, Oslo, Norway.

Mark M, Ghyselinck NB, Chambon P. (2006). Annu. Rev. Pharmacol. Toxicol.46, 451-480.

Miettinen, H.M., Pulkkinen, P., Jamsa, T., Koistinen, J., Simanainen, U.,Tuomisto, J., Tuukkanen, J. and Viluksela, M. (2005). Toxicol Sci 85, 1003-12.

Moverare, S., Venken, K., Eriksson, A-L., Andersson, N., Skrtic, S.,Wergedal, J., Mohan, S., Salmon, P., Bouillion, R., Gustafsson, J-A.,Vanderschueren, D., Ohlsson, C. (2003). Proc Natl Acad Sci USA 100, 13573-8.

Nilsson CB and Hakansson H. (2002). Crit Rev Toxicol. 32(3), 211-32.

Olausson, H., Herlin M., van der Ven, L., van der Kuil, A., Verhoef, A.,Leonards, PEG., Piersma, AH., Vos, JG., Håkansson, H. (2006). In: Dioxin2006, Oslo, Norway.

Pastorelli, R., Carpi, D., Airoldi, L., Chiabrando, C., Bagnati, R., Fanelli, R.,Moverare, S. and Ohlsson, C. (2005). Proteomics 5, 4936-45.

Schmidt, C.K., Volland, J., Hamscher, G. and Nau, H. (2002). BiochimBiophys Acta 1583, 237-51.

Figure 2. Rat primary bone cells grown on bovine bone slice and visual- ized by confocal microscopy. AhR is stained red by rhodamine conjugated secondary antibody, and F-actin is demonstrated by fluoresceinisothiocyanate (FITC)-conjugated phalloidin. The actin ring containing multinuclear cell is an osteoclast while the surrounding cells are osteoblasts. Scale bar is 50µm. (Image by Joanna Ilvesaro)

BONETOX



In vitro profilingWe finally studied 16 plasticisers and ‘orphan phenols’ (phe-nolic compounds believed to have ED activity in vivo, such aso -phenylphenol) and used 23 in vitro tests. Cluster analysis ofthe results, linked with those from our genomic/proteomicarrays, has shown that ED activity can be predicted using arelatively small number of tests. We are currently aiming topatent this to provide in vitro profiles which could be used inidentification of compounds as EDs. This development of invitro tests would reduce the use of animals in reproductivetoxicology and would potentially be compatible with therequirements of the REACH programme.

Members of the ENDOMET consortium,

Rosemary Waring, David RamsdenUniversity of Birmingham, UK

Christian BehlJohannes Gutenberg-Universitat, Germany

Eva Bonefeld-JørgensenUniversity of Aarhus, Denmark

Ulrich LoosUniversity of Ulm, Germany

Maria Fickova and Sona ScsukovaInstitute of Experimental Endocrinology, Slovakia

http://endomet.bham.ac.uk/

ENDOMET

The ENDOMET project Dysregulation of ENDOgenous steroid METabolism potentially alters neuronal and reproductive system development: effects of environmental plasticisers was set up to determine whether environmental plasti-

cisers (EPs) could interact with human neuronal and repro-ductive systems. It was also envisaged that the programmewould provide in vitro tests, largely using human cell lines,which could be used to identify potential endocrine disrupters(EDs). Now that ENDOMET has finished (June 2006), theresults from all the groups are being summarised and collat-ed to give a series of key achievements and questions.

Non-genomic pathwaysEDs are classically described as acting as agonists/antago-nists at steroid receptors, the so-called ‘genomic pathway’.Although this certainly occurs, the mode of action of manyEPs, especially the phthalates and alkyl phenols, can best be

explained by their effects on steroid sulphation, steroid syn-thesis, cell signalling and cell transport. In particular, phtha-lates, which in vivo have major endocrine-disrupting potential,have been shown to act on this non-genomic basis.

Tissue specificityEven when genomic aspects are considered, there appearsto be tissue specificity: although human neuronal cell linescontain oestrogen and androgen receptors, they are relative-ly insensitive to these ‘genomic’ effects of EPs (apart fromthose of bisphenol-A), unlike the human reproductive celllines.

Neuronal enzymesSome EPs affect the neuronal enzyme dehydroepiandros-terone (DHEA) sulphotransferase (SULT 2A1) which convertsDHEA and pregnenolone into their corresponding sulphates.This process is known to be essential for memory formationand retention. Many EPs are fat-soluble. If they were notmetabolised, could they cross the blood brain barrier, accu-mulate and then have subtle effects over a lifetime on mem-ory and cognitive function? At present we do not have theanswers but this seems a possibility, particularly as EPs canalso affect neuronal cell signalling.

Foetal development

Many EPs affect the thyroid system and also alter iodideuptake into the cell, so indirectly modulating thyroid function.As the thyroid is critically important in neurodevelopment,could EPs accumulate in the foetus and alter brain structureand/or function?

What have we learnedfrom ENDOMET?

Selected publications

Many of the results from ENDOMET have been published ina special issue of Molecular and Cellular Endocrinology (MCE) , December 2005 volume 244 (1-2), see below.

Ghisari M and Bonefeld-Jorgensen EC, Impact of environmental chemicalson the thyroid hormone function in pituitary rat GH3 cells, MCE 244, 31-41,2005.

Waring RH and Harris RM, Endocrine disrupters: a human risk?, MCE 244, 2-9, 2005.

Harris RM, Kirk CJ and Waring RH, Non-genomic effects of endocrine dis-rupters: inhibition of estrogen sulfotransferases by phenols and chlorinatedphenols, MCE 244, 72-4, 2005.

Turan N, Waring RH and Ramsden DB, The effects of plasticisers on 'sul-phate supply' enzymes, MCE 244, 15-9, 2005.

Wenzel A, Franz C, Breous E and Loos U, Modulation of iodide uptake bydialkyl phthalate plasticisers in FRTL-5 rat thyroid follicular cells, MCE 244, 63-71, 2005.

Breous E, Wenzel A and Loos U, The promoter of the human sodium/iodidesymporter responds to certain phthalate plasticisers, MCE 244, 75-8, 2005.

Mlynarcikova A, Kolena J, Fickova M and Scsukova S, Alterations insteroid hormone production by porcine ovarian granulosa cells caused bybisphenol A and bisphenol A dimethacrylate, MCE E 244, 57-62, 2005.

Mlynarcikova A, Fickova M and Scsukova S, Ovarian intrafollicular process-es as a target for cigarette smoke components and selected environmental

reproductive disruptors, Endocrine Regulations 39 (1), 21-32, 2005.



EASYRING

Rapid non-invasive testing methodsEASYRING final report

Endocrine disrupting chemicals (EDCs) can cause adverseeffects on the reproductive biology of vertebrates. Thesecompounds may have a marked impact on reproductive biol-ogy by mimicking or antagonising the effects of naturallyoccurring sex steroids or other natural hormones, such as via(anti)oestrogenic and (anti)androgenic action. Starting fromthis knowledge, the project Environmental Agents SusceptibilitY assessment utilizing existing and novel biomarkers as Rapid non-invasive testING methods (EASYRING)undertook its research programme as a true ‘ring’. The proj-ect moved full circle, starting from the environment and theRiver Lambro (the most polluted tributary of the River Po,Northern Italy), by means of in vitro and in vivo studies andmathematic models, to improved information on the environ-mental levels of some known EDCs and their biologicaleffects on reproduction. Several specific tasks were under-taken to meet these broad aims, both in aquatic species (thecommon carp, Cyprinus carpio, and the African clawed frog,Xenopus laevis, as models) and in small mammals (mice).This has generated large amounts of multidisciplinary data,much of which has already been or is due to be disseminat-ed in scientific papers and at international meetings. Thesetasks were successfully completed at the conclusion of theproject, in December 2005.

River contaminationFocussing on the aquatic environment, moderately low levelcontamination of the River Po and particularly of the middlesection were documented by chemical analyses of watersamples collected upstream and downstream at the conflu-ence of the River Lambro. By contrast, the results of sedi-ment and macro invertebrate analyses suggested a clear dis-tinction between the upstream and downstream stretches ofthe middle River Po, showing the consequences of EDCloads from its polluted tributary. The discrepancy betweensediment and overlying water is not surprising, and can beexplained by the moderate lipophilicity of EDCs and the high-er recording capacity of bed sediments compared to grabwater samples. Furthermore, fish sampled downstream atthe confluence of the River Lambro showed a trend towardsincreasing plasma levels of vitellogenin (VTG), the confirmedbiomarker for oestrogenicity, altered steroid plasma levelsand morphological alteration of gonads and liver.Consequently, the first step of EASYRING was to use differ-ent in vitro assays (two recombinant stable human cell lines,MVLN and MDA-kb2, in parallel with the yeast oestrogenscreen (YES) and the yeast androgen screen (YAS) as a pre-liminary screening of the water and sediment fractions inorder to gain more knowledge about the presence of EDCs.The screening revealed the presence of a mix of compoundswith different mode of actions, namely oestrogenic, andro-genic and anti-androgenic compounds. Information fromtests consistently identified a small number of fractionsas being mainly responsible for oestrogenicity. Chemicalanalyses undertaken on these fractions identified a smallnumber of known oestrogenic chemicals, i.e. oestrone (E1),17 β-oestradiol (E2), oestriol (E3), nonylphenols mix (NPs),

bisphenol A (BPA), and t -octylphenol (tOP). The three naturaloestrogens and the three industrial chemicals identified in thehighly oestrogenic fractions were chosen to be used in the‘environmental mixture’ (1x Lambro) for in vivo exposures ofcommon carp and X. laevis .

With this premise and with the aim of interpreting the alter-ations found in feral fish of the River Po, the effects of grad-ed concentrations of the most interesting of the above-men-tioned oestrogenic chemicals as well as of their mixture,were examined during the second year of the project in sucha way as to mimic environmentally relevant levels and com-positions. Early development of gonads and sexual differenti-ation are very sensitive to hormonal imbalance and thereforeto the action of those chemicals which can affect the com-plex functions of the reproductive system. To investigatethese processes, and specifically how the River Lambro mayaffect them, carp fingerling which had undifferentiatedgonads and still under development, were exposed to the 1xLambro mixture for five months. The study was completedby two other treatments in which carp fingerlings wereexposed to River Po bed sediments, collected upstream anddownstream of the confluence of the River Lambro. In thisway, carp were also exposed to the ‘natural’ mixtures ofEDCs present in the main Italian river particularly after the netenrichment transported by the River Lambro. Alteration ofgonad differentiation, gonad morphology, sex ratio andsteroid ratio and plasma VTG induction were indeed observedin carp juveniles exposed to environmental mixtures anddownstream sediment.

Dipstick assayA specific goal of EASYRING was to develop a new non-inva-sive system for the easy detection of VTG directly in the fieldin order to assess oestrogenic responses in the carp, a com-mon fish found in European waterways and regularly used inenvironmental monitoring programmes across Europe. Ourdata showed that VTG can be detected in fish mucus using alateral-flow immunoassay (LFIA; dipstick assay) performedwithin a few minutes of sampling, at VTG concentrations lowenough to detect oestrogenic effects in the environment. Toverify the applicability of the dipstick tool and its related pro-tocol, the participants operated in the presence of personnelfrom ARPAL, the Regional Environmental Protection Agency.Comparison of VTG levels in plasma and mucus of the samefish using a quantitative sandwich ELISA previously estab-lished indicates a positive correlation in almost all groups ofcarp investigated. However, mucus VTG in some fish groupsdid not reflect plasma VTG as linearly as observed in morecontrolled laboratory studies, and care should therefore betaken when using the non-invasive strategy in the field.Apparently, exposure to anti-oestrogens may disturb the VTGturnover such that mucus and plasma VTG are no longer inequilibrium. Possibly, a blood sample should be taken, whichcould be analysed by the LFIA in a few minutes in the field.

From larger specimens, blood sampling could also be a non-destructive strategy. Further studies should be performed toelucidate this.

Alberta Mandich University of Genoa, Italywww.easyring.org




Twenty years of basic research tobe translated into more predictive

risk assessmentEndocrine disrupters (EDs) are environment and food con-taminants known to alter metabolic functions of higher ani-mals by interfering with the activity of a specific class ofreceptors: the intracellular receptors (IR). Because a signifi-cant number of man-made compounds show endocrine dis-rupting properties, regulatory agencies are recognizing theimportance of defining acceptable limits of ED concentrationsin the environment and are starting to discuss the extent towhich currently available methods are applicable to riskassessment of ED.

Thanks to in-depth investigation carried out on the IR mecha-nism of action over the last twenty years, we now know thatIRs are finely regulated transcription factors controlling theactivity of selected promoters in concert with general or cellspecific co-regulators (co-activators and co-repressors). Theinteractions with co-regulators explain why synthetic IR lig-ands were found to exert cell- and promoter-specific action.Application of molecular biology tools revealed that IRs tar-geted by EDs are present in most, if not all, cells in mammals,and are profoundly involved in the control of most metabolicfunctions, pointing to the widespread undesired effects thatED might exert. Thus, in view of the newly acquired aware-ness on IR mechanism of action and functions there is a

growing concern on the validity of the methodologies so farapplied to identify EDs and to predict their harmful effects.Indeed, current in vivo and in vitro methodologies restrict theanalysis to very specific organs or cell types and, therefore,are not sufficient to predict the potential consequences ofEDs after short or long-term exposure. The recent emphasison generating in vitro model systems for toxicological analysishas certainly discouraged the development of models that aresuitable to envision the whole spectrum of effects on thebody, which is required when dealing with endocrine disrup-tion that is, by definition, a living organism.

Novel opportunities

Molecular imaging techniques applied to animal engineeringnow provide novel opportunities to create model systems thatportray the systemic activities of a specific molecule in realtime by means of non invasive technologies. For the firsttime, this enables measurement of the biological activity of agiven compound without pain or sacrifice of the laboratory ani-mal. These models are particularly suited to the study of tran-scription factors, such as IR, because of the facility to assessthe expression of genes encoding bioluminescent or fluores-cent proteins, the so-called reporter genes. Typically, areporter system for the study of IR-dependent transcription isgenerated by integrating into the mouse genome a hormone-

responsive promoter driving the exogenous reporter gene(figure 1). With the aid of European funds (BRIDGE-BIOT-CT92-0308), our laboratory planned and produced a first para-digmatic reporter mouse, with the ERE-Luc that providesmajor insights on ER physiology.

The EDERA programme was initiated with the aim of testingthe suitability of the ERE-Luc mouse as a novel model systemto: i.) identify presence of oestrogenic compounds in food andenvironment; ii.) provide a complete pattern of the body dis-tricts perturbed by xenoestrogens; iii.) assess the potentialrisk of acute or chronic exposure to xenoestrogens.

Bioluminescence imaging

EDERA’s main task was to generate protocols based on invivo bioluminescence imaging and demonstrate their applica-bility to toxicological and risk assessment analysis. This maingoal was achieved by means of a series of more specific

tasks. With comparative studies involving the quantitativeanalysis of photon emission in vivo and luciferase enzymaticactivity ex vivo we could demonstrate that imaging is amethod applicable to obtain a map of the effects of differentconcentrations of a given xenoestrogen in space and time (fig-ure 2). The faithfulness of luciferase as reporter of ER tran-scriptional activity was shown by comparing the effect ofadministration of oestrogens of different origin (the naturalhormone 17 β -oestradiol; the phytoestrogen genistein and thesynthetic oestrogens p ,p ’-DDT and BHC) on the accumulationof luciferase or transcripts from endogenous ER target genes(such as progesterone receptor, CYP17 and the oestrogenreceptors themselves).

In a second subtask, the imaging-based methodology wasapplied to the study of the effects of long-term exposureto food oestrogens like the phytoestrogen genistein orto complex mixture of oestrogens such as soy milk. Our

EDERA

Figure 1: Reporter mice for in vivo studies of receptor activity. The ERE- Luc reporter mouse was generated by the integration of a transgene carrying the firefly luciferase gene under the control of a duplicated oestrogen responsive element (ERE) located upstream of a minimal thymidine kinase gene. Insulator sequences prevent any influence of the surrounding chromatine on the synthetic transgene. When the substrate luciferine is made available, the luciferase enzymatic activity results inthe production of photons which can be detected by the use of a CCD camera and with the aid of specific algorithm that derives the intensity of the photon emission in each area of the mouse an image can be generated. An appropriate scale of colours can be generated in order to visualize the intensity of photon emission.

November 2006 www.credocluster.info



experiments showed, for the first time, that phytoestrogensaccumulate in the body and in long-term exposures interferewith ER signalling in a tissue selective manner. Most interest-ingly, treatment with soy milk generates a state of activationof ERs which differs significantly from that which was shownwith genistein: in long-term treatment we observed a slightactivation of ERs in liver, but a very significant activation intestis, indicating that the pure substance, genistein, may haveeffects significantly different than mixtures of phytoestrogenseven if enriched in genistein itself. The model system provedparticularly sensitive to exposure to different concentrationsof xenoestrogens because it could show different responsesto administration of pure or dilute soy milk and the state of ERactivity was directly proportional to the amount of soy milkingested. These data point to the necessity to better investi-gate the potential ED activity of soy milk, particularly whenadministered to infants of both sexes. Another EDERA sub-task tested the possibility to generate a reporter systemdiscriminating ER subtype specific oestrogens. To this aim,we bred the ERE-Luc mouse into the BERKO mouse.

Preliminary results, to be further confirmed, demonstratedthe validity of the system in which luciferase is activatedonly by ER α specific compounds. The last subtask of theEDERA project addressed the construction of novel reportervectors for the generation of reporter animals to be usedto identify and study ED acting via androgen and thyroidhormone receptors.

Replacing, reducing and refining

Overall we feel that the outcome of the EDERA project is verypositive in terms of environmental and food protectionbecause it has clearly shown the major advantages associat-ed to the use of reporter animal technology with respects tothe currently available model systems. In our view, thereporter mouse technology is an excellent candidate toREPLACE the existing tests that, for their nature, are unableto provide an overall view of oestrogenic activity in the wholeorganism. By means of non-invasive in vivo imaging technol-ogy, the methods provide the opportunity to REDUCE thenumber of animals to be used in the in vivo tests first of allbecause animal sacrifice is not needed, and secondly the pos-sibility to follow the endocrine effects in time in the same ani-mal reduces the need to use large numbers of animals in eachexperimental group to reach significant data and also reducesthe need for control groups (the effects of a treatment is eval-uated versus the baseline state of activity of the receptor onthe same animal). Last, but not least, the technology willREFINE current methods by providing for the first time thepossibility to study the effect of EDs systemically and, afterlong-term exposure even to low doses, our methodology willeliminate the pain created by animal testing and abolish thenecessity of animal sacrifice.

Thus, we feel that the work carried out by the EDERAconsortium has shown the advantages of novel transgenicanimal models for the study of EDs effects and exposure. Thestudies carried out with the ERE-Luc mouse need to beextended to generate other reporter systems, perhapsenabling study of the activity of more than one IR in the sameanimal. Studies along these lines are currently being carriedout in new consortia under the Sixth Framework Programmefor Research and Technological Development. Within the NoECASCADE, EMIL and DIMI we are collaborating in the gener-ation of novel, bimodal imaging vectors and new reporter ani-mals; within the STREP EWA and the IP CRESCENDO we arepursuing in the investigation of the physiological changes ofER activity during development and aging; and within theSTREP EXERA we will collaborate with several SMEs to testthe feasibility of the use of reporter mice for the generation of3d cultures of specific target tissues for high-throughputanalysis of EDs.

Adriana MaggiUniversity of Milan, Italy

www.edera.unimi.it

Figure 2: Time course analysis of p,p’-DDT activity in ERE-Luc mouse.

Male ERE-Luc mice fed with oestrogen-deprived diet were injected with50 µg/kg of 17 β-oestradiol or with p,p’-DDT and photon emissionanalyzed at different times. The upper panel shows a typical portray of the image generated by the CCD camera is shown. In the lower panel is the semi-quantitative analysis of the photon detected in selected body districts.


EDERA



The mechanisms of action of endocrine disrupters (EDs) arenot well known either at the molecular, cellular, tissue ororganism levels, or during the critical periods of development.EDs may act as endocrine deregulators and generate genederegulation depending on the genetic background of theorganisms. Consequently, analysis of the genetic effects ofthese compounds on developing gonads could facilitateassessment of the mechanisms of action of EDs upon theindividuals and the progeny of future generations by genetictransmission.

The mammalian testis and the biological process of cell dif-ferentiation to spermatozoa (spermatogenesis) have beenidentified as targets of special interest, due to several reportsand epidemiological studies showing increases in male infer-tility and testicular cancer incidence. Our research aimed toevaluate EDs and their effects on animals and humans duringdevelopment, including fetal and neonatal periods.

A genetic approachThe GENDISRUPT project took a genetic approach andfocused analysis on the effects on, and the genetic suscepti-bility of testicular cells to selected endocrine disrupters. Twomain models were used for this: mouse and human. Themouse model was based on both in vitro and in vivo approaches. The in vitro approaches were carried out onprimordial germ cells (PGCs) and Sertoli cells. The in vivo approaches, based on mice, were designed such that expo-sure of mothers in pre-mating period could show epigeneticeffects, exposure during embryonic development to indicateprenatal effects, and exposure during the pubertal period todefine effects during puberty.

Several compounds with reported oestrogenic activity weretested in the mouse model:• Bisphenol A (BPA), which is a component of polycarbonate

plastics and is a contaminant possessing a weak oestro-genic activity.

• Lindane, the gamma isomer of hexachlorocyclohexane(HCH), is one of the oldest synthetic pesticides still in useand affects reproductive function in animals: decreasedspermatid numbers and Sertoli cell fragmentation havebeen reported.

• A phthalate, mono(2-ethylhexyl)phthalate (MEHP), thetoxic metabolite of the widespread plasticiser componentdi(2-ethylhexyl)phthalate (DEHP). Phthalate esters werefound to cause malformations in the reproductive tract ofintra-uterine exposed males.

• Zearelanone (ZEA), a nonsteroidal phytoestrogen from thegroup of resorcylic acid-lactones has been reported tocause malformations in the reproductive tract and germcell apoptosis in the testis.

• 17 β-oestradiol (E2) was also studied, as a natural oestro-genic compound.

Key objectivesThe main objectives of this work were to undertake:1. selection and characterisation of genes showing deregula-tion of expression, by the action of EDs, during the develop-ment and differentiation of mouse testicular cells;2. a comparative analysis of gene deregulation in relation totreatments, dosage and cell types affected; and3. phenotypic evaluation of the effect of EDs in mouse testisdevelopment.

The analysis of gene expression was mainly carried out oncommercial microarray platforms which contain most of themouse transcriptome. This approach allowed us not only todefine molecular markers for gene expression deregulationas an effect of EDs in testis development, but also to definequantitatively the effects of exposure period and dose. Wecould also address specific effects of EDs, such as "low doseeffect" or the emerging question of epigenetic effects. In par-allel, a specific prototype of microarray (GENDISRUPT-1) con-taining 300 specific oligonucleotides (from genes selected ina previous EC project, BIO4-CT96-0183) was also designedand evaluated.

Subsequent studies were undertaken using functionalgenomics and proteomics approaches. The combination ofboth approaches can facilitate the understanding of gene reg-ulation (and deregulation) and the potential effect of genederegulation in the cells and biological systems under study.Detailed comparative anatomical analysis, testis histopathol-ogy and hormonal status were carried out on the same sam-ples from ED-exposed mice that were evaluated for geneexpression deregulation.

The developing gonadAlthough the reproductive system is considered a main targetfor EDs, scant information is available on the effects ofoestrogens and EDs on the development of embryonicgonad, and in particular, of PGCs. In the mouse, several con-ditions are known to influence PGC development and toresult in sterility or development of germ cell tumours. Todate, there have been few studies which have included athorough investigation of the expression of oestrogen recep-tors (ER α and ER β) in gonadal somatic cells and germ cellsduring the crucial period of PGC development.

Human studiesWith regard to the human studies, an aim of this project wasto identify molecular markers that might explain genetic sus-ceptibility to endocrine disruption in humans, focusing onmale infertility and testicular cancer. These two traits arethought to be complex diseases in which the interactions ofenvironmental (i.e. EDs) and genetic susceptibility factorscould condition their development. The human studies werefocused on clinical cases of testicular carcinoma and infertili-ty. Analysis of single nucleotide polymorphisms (SNPs) of tar-

get genes and linkage analysis of quantitative trait loci (QTLs)can address the genetic susceptibility to EDs. Data miningprocessing and high-throughput genotyping methods haveenabled some gene associations with male infertility to beestablished.

Genetic markers in testisendocrine disruptionAchieved and achievable

November 2006 www.credocluster.info

GENDISRUPT



Genetic associations with male infertilityNineteen candidate genes were selected for genetic associ-ation studies of human male infertility and testicular cancerand 46 SNPs were selected to perform these studies.However, only 37 of these SNPs were validated in our popu-lation and high-throughput genotyping protocols weredesigned for these 37 polymorphisms.

To perform the genetic association studies, we recruited atotal of 167 DNA samples from idiopathic infertile men, 1000DNA samples of people from the general Spanish population,101 DNA testicular tissue samples from testicular cancerpatients, and 7 testicular tissue samples from healthy donors.To perform these analyses, a total of 28 SNPs were evaluat-ed within eleven candidate genes and a total of 13,543 geno-types were obtained. The findings support a genetic basis ofhuman male infertility and we observed associations of theESR1, KIT, KITLG and PTEN genes with male infertility. Theseobservations are in accordance with the possible existence ofmale infertility susceptibility factors within these genes inhumans. An important finding is the implication of the ESR1gene in male infertility. It is interesting to note that theseresults have been also observed in three independent seriesfrom Greece, Japan and Italy.

Multilocus analyses of FSHR, ESR1, ESR2, CYP19A1 andNRIP1 revealed the existence of genetic interaction betweenthese genes and their possible implication in the human maleinfertility. Preliminary evidence of loss of heterozygosity inthe PTEN gene has been detected in human testicular cancer.Further studies of this gene in a larger group of testicular can-cer patients could reveal if this observation can be consideredas a common event in testicular cancer pathology.

Microarray studiesThe results of mouse studies using microarrays wereobtained by analysis of more than 3 million individual genedata. This represents an enormous effort of analysis neverdone before to evaluate comparative gene expression dereg-ulation induced by EDs in developing testis. As a conse-quence of this analysis, which is still in progress, we have thefollowing observations to report:• MEHP is the ED which has demonstrated the strongest

effect, based on the highest level of gene deregulation;• MEHP and ZEA exposure define specific gene expression

signatures by microarray analysis;• The highest deregulation effects, for all the EDs analysed,

was on mice that were exposed to the different EDsthroughout all life stages up to analysis at 4 weeks post-natal;

• However, MEHP also induced remarkable deregulationwhen it was given to mothers during the embryonic peri-od, in spite of the fact that animals were not exposed dur-ing the pubertal period.

• In general, E2 showed lower effects on gene deregulation

in testis than the other EDs tested.• A ‘low dosage effect’ was evident in the longest period ofexposure to MEHP and lindane.

• A proteomic profile of the soluble proteins expressed atdifferent stages of mouse testis development was carried

out and 42 proteins were identified. Conspicuous varia-tions in their accumulation (representing up or down-regu-lation) were detected.

In general it may be concluded that the effects of different EDsanalysed during testis development do not follow the samepathways at the level of gene deregulation. Furthermore, mostof the EDs analysed in this study have a pattern of gene dereg-ulation which is distinctive from that of E2.

Morphologically visible effectsStudies on the possible effects of ED treatments on mousetestis development also revealed some morphologically visi-ble effects on the seminiferous epithelium, including espe-cially higher numbers of apoptotic cells and of diploid sper-matids.

Results obtained from the study of PGCs provide the firstindication that exposure to oestrogens during embryonic lifemay have profound effects on germ cell growth and differen-tiation through the action of gonadal somatic cells and themolecular pathways identified. Embryonic exposure to highlevels of oestrogens or EDs constitutes a risk for PGC trans-formation in pluripotent tumorigenic cells. This process couldbe at the origin of germ cell tumour formation in the testis.

An in vitro assay has been set up to identify and quantifyoestrogenic effects on fetal testis cells. After validation, thisassay might represent the basis of a novel test for rapidoestrogenic and anti-oestrogenic activity screening and inves-tigation into the molecular pathways underlying the effect ofthese compounds on fetal gonads.

ConclusionsFrom these studies, it is apparent that the complex mecha-nisms of endocrine disruption could be involved in testisdevelopment. The type of compound, the dose, and thedevelopmental stage of exposure could operate on differentgene expression patterns and consequently affect differentmolecular pathways. Establishing a defined pattern of geneexpression associated with specific compounds and withmixtures of potential EDs will be one way to design and vali-date analytical tools based on microarrays and new technolo-gies in development. GENDISRUPT has contributed to theapplication of genetic analysis to identify molecular markersand in the knowledge gained on the mechanism of action ofenvironmental chemicals on animal and human reproduction.Further studies will support and enhance the results obtainedin GENDISRUPT towards controlling the adverse effects ofendocrine disrupters and other reprotoxicants.

Jesús del Mazo, Pedro P. López-Casas and María PazDepartment of Cell Biology and Development, Centro deInvestigaciones Biológicas (CSIC), Spain

http://gendisrupt.cib.csic.es/

Theimageinthetopbarshowsapartialviewofmicroarrayanalysis.



Many international organisations, such as the EuropeanCommission, the Organisation for Economic Co-operationand Development and the World Health Organisation, as wellas national governmental departments, such as the USEnvironment Protection Agency, have launched cooperativeinternational activities to test and assess thousands of syn-thetic chemicals found in many common products for theirpotential effect on the hormonal systems of humans and ani-mals in order to address reproduction related disorders. Themain conclusion of most reports resulting from these activi-ties up to now was the urgent need to establish reliable testmethods. Both in vivo assays and in vitro screening systemsare required to assess the endocrine disrupting potential ofchemicals and to test for the interference of compounds withthe oestrogenic and androgenic pathways or thyroid system.

The general goal of the MENDOS project on biomimetic opti-cal sensors for environmental endocrine disrupter screeninghas been to provide novel analytical technology for screeningendocrine disrupting chemicals (EDCs), both in terms of cost-effective tools for rapid pre-screening of environmental sam-ples as well as clarification of potential EDCs modes of actionand risk assessment. The project has focused on developingnovel biomimetic recognition materials based on molecularlyimprinted polymers (MIPs) together with highly sensitiveminiaturised optical transducers. In addition, alternativeapproaches based on whole cell-based biosensors and bioas-says as well as DNA microarray technology have also beenfollowed.

Due to the large number of chemicals with potentialendocrine effects, screening methods should be capable ofdetecting many classes of compounds in a reasonably shorttime (‘high throughput screening’) and offer the possibility ofon-site analysis to identify sources of contamination in sur-face waters and sources of drinking water. Current technolo-gies barely accommodate these requirements since they relyalmost exclusively on biological/biochemical assays, whichsuffer from the general disadvantages of biosensors/bioas-says, including sophisticated preparation and handling, lack ofstability, use of (radio)labeled components or biomarkers, lim-ited storage capability and subsequent high costs.

Almost all of these drawbacks might be avoided by the intro-duction of novel biomimetic screening systems based onintegrated optical sensor technology, as a non-animal artificialreceptor-based fast screening method for environmentalEDCs. The combination of optical sensor technology and arti-ficial biorecognition layers mimicking the biofunctionality of areceptor/antibody with the advantageous properties of poly-mers should be designed to be applicable in environmentalmonitoring schemes. However, as no single test can assessall potential EDCs in the environment, the MIP sensors weredeveloped in comparison to new biosensor systems basedon cellular systems. While the MIP sensor should identifyEDCs by their structure, the bioassays should respond to abroader range of EDCs by measuring cellular effects andallow detection of EDCs in complex samples with relevance

for hazard and risk assessment to the environment and ofhuman health.

Biomimetic recognition materialsSeveral main technological developments in the field of bio-mimetic and biological sensor systems for EDCs wereachieved during the 3-year MENDOS project. The studies onbiomimetic recognition materials resulted in the developmentof MIPs which selectively could recognize priority pollutantssuch as 17 β -oestradiol, benzo[a]pyrene (BAP), diethyl-hexylphthalate and atrazine. These MIPs were obtained bothin bulk format as well as in the form of micro- or nanospheresuseful for application in binding assays or as stationary phas-es in chromatography or solid phase extraction (SPE). Next tothe selectivity, MIP development studies have consideredparticle size for the respective applications. With the propersynthetic strategy, the size and morphology of the imprintedspheres can be rationally controlled by varying the polymeri-sation conditions, the nature of the cross-linker, the monomerconcentration and the polymerisation temperature (figure 1).For chromatographic evaluation (e.g. HPLC) or SPE, MIP par-ticles with dimensions ranging from 10 to 25µm are usuallyapplied, compared to binding assays with MIPs immobilisedat a chemical sensing interface where smaller particles (lessthan 1µm) with a narrow size distribution are required. Themain advantage of using MIPs as synthetic sorbents withantibody-like binding properties is their biomimetic recogni-tion ability, which enables them to selectively extract one orseveral structurally related analytes at trace concentrationsfrom complex environmental mixtures – the separation ofoestrogens from natural organic matter was obtained in asimple two-step extraction procedure. In addition, MIPs arestable under harsh chemical conditions, so that a wide varietyof binding and eluting reagents can be applied without loss ofrecognition performance. It has furthermore been demon-strated that the developed MIP sorbents favourably compareto commercially available C18-based SPE materials in termsof selectivity and recovery, providing an advanced samplepreparation tool for selective extraction of oestrogens fromcomplex aqueous samples (figure 2).

MENDOS

Figure 1 (above left): Scanning electron micrographs (SEM) of imprinted micro and nanospheres (images provided by B. Mizaikoff; published inWei et al. Biosens. & Bioelectr. 21, 1943-1951 , 2006).

Figure 2 (above right): Total ion chromatogram (TIC) and selected ionchromatogram of spiked river water at 17 β -oestradiol 50 ng/L after MIP based SPE (images provided by B. Mizaikoff; published in Wei et al.Biosens. & Bioelectr. 21, 1943-1951 , 2006).

Developing biomimetic and biological sensor systems for EDCs



Similarly, molecularly imprinted polymers in the form of bulkpolymers or microspheres for the target analyte BAP weresuccessfully prepared using 4-VP and DVB as functional andcross-linking monomers. The resultant MIPs showed goodrecognition towards BAP over the nonimprinted polymerswhile other tested polyaromatic hydrocarbons showed signif-icantly reduced binding.

MIP films were also obtained by assembly of MIP-nanos-pheres via linear-polymer interlayers. The advantage of thiscoating technique is that it is reversible, that it can be done insitu even in a flow-cell, and that it can be done with substrateshaving a high aspect ratio, that are difficult to coat by spin-coating. Using atrazine imprinted particles in surface plasmonresonance (SPR) mode, it has been shown that the layers didrespond to the presence of the analyte.

Optical sensor technologyIn terms of optical sensor technology, both novel interfero-metric transducers as well as SPR based optical sensingplatforms have been developed. In particular, ( i.) a mobile SPRsensor based on prism coupling, spectral modulation andwavelength division multiplexing and ( ii.) a high-throughputSPR system based on angular spectroscopy of surfaceplasmons on an array of diffraction gratings (figure 3). TheSPR sensor method was combined with antibodies andMIPs for detection of selected EDCs. Antibody-based SPRsensors detected selected EDCs (e.g. atrazine, 2,4-dichloro-phenoxyacetic acid, BAP, and 4-nonylphenol) in sub-ng/mlconcentrations.

Biosensor systemsIn parallel, biosensor systems were developed in order to getvalid screening methods with biological relevance. A whole-cell bioassay, consisting of genetically modified eukaryoticcells using a mammalian vector carrying a Luc-reporter gene,under the control of the Drosophila melanogaster hsp22stress inducible promoter was developed. This assay wasshown to give a sensitive signal, dependent on both toxicityand bioavailability of the tested compounds – such as heavymetals, genotoxic agents and endocrine disrupters. By opti-misating culture of the human H295R cell line and a non-radioactive detection method (ELISA), an aromatase assaywas developed as a test system for the interference of xeno-biotic chemicals with steroid metabolism. This specific end-point, in addition to the interference with hormone receptors,is of high relevance to be included in the endocrine disruptiontest strategy. Furthermore, a system for assessing the oestro-genic activity of a sample in situ has been established basedon immobilised chemo-sensitive luminescent yeast cells. Itwas demonstrated that luminescent yeast based hydrogelbioassays are applicable to the analysis of environmentalwater samples, potentially as a primary screening tool and asthe biological and chemical immobilisation parts in the con-struction of fibre-optic biosensors. The possibility of long-termstorage, easy and cost-effective preparation, no need for con-tinuous cell cultivation, and only 2.5 hours assay time, makethe immobilised oestrogen inducible yeast-based hydrogel apromising alternative to the recombinant yeast reporter geneassays that are currently used.

DNA chips for detection of EDCsFinally, DNA chips holding oligo DNA probes for hormoneresponsive genes were successfully developed for the detec-tion of EDCs. Several dedicated chip surface chemistrieswere applied and evaluated for quality and performance char-acteristics. The epoxy surface showed to be most appropri-ate for the effective binding of NH2 modified 50bp longoligonucleotides. The up and down regulation of the oestro-gen regulated genes by EDCs could be detected in ZR75breast cancer cells using this oligo DNA chip. Detection ofanti-androgenic action of EDCs has been feasible utilizing22RV prostate cancer cells. While the identification of indi-vidual EDCs is not possible in environmental samples, itcould be shown that DNA chip-based assays are able toreveal the presence of oestrogenic compounds based ontheir hormonal activity even in low concentrations.

The main objective envisaged for the MENDOS project wasthe development of field-deployable monitoring systems forEDCs in the aqueous environment. Although it has not yetbeen possible to establish a fully functional MIP-based opticalsensor platform for EDC monitoring, significant advanceshave been made in MIP technology and optical sensor equip-ment, thus setting the basis for successful future implemen-tation of highly sensitive biomimetic optical sensors. The lat-ter, as well as biosensors and DNA chips could, after furtherrefinement and validation, be used as tools to screen foroestrogenic activity of environmental samples or chemicaltest compounds, which is of relevance for monitoring waterquality (Water Framework Directive) and chemical safety(REACH).

Hilda WittersVITO (Flemish Institute for Technological Research),Environmental Toxicology Unit, BelgiumMichael JakuschARC Seibersdorf research GmbH, Austria

www.mendos.org


MENDOS

Figure 3: A laboratory prototype of a high-throughput SPR sensor based on spectroscopy of surface plasmons on an array of diffraction gratings (left) and detail of the sensor chip (right) (development at IREE, images kindly provided by J. Homola).



IntroductionThe European Commission’s Environment and Health (E&H)Action Plan, adopted in 2004, has as its main aim to improvethe understanding of the link between environmental factorsand health. The implementation of the goals of this actionplan through research has started in FP6 via funding of sev-eral large- and small-scale research projects on topics identi-fied as priorities in the Action Plan. In FP6 a new approachwas introduced for integrated environment and health riskassessment methodologies. FP6 projects such as NOMIRA-CLE or INTARESE will examine the concepts towards inte-grated risk assessment and link the environment and healthdata in a more effective way to also better support environ-ment and health-related policy making. Along this line, theconcept ‘full-chain approach’ was introduced, putting moreemphasis on the identification of pollution sources, expo-sures (including human biomonitoring), links to health effects,and economic valuation. The third new concept in FP6 dealtwith the economic valuation models and tools to be devel-oped for the area of environment and health in order to sup-port impact assessment.

The efforts will continue and intensify during FP7. Althoughnew knowledge has been or is being generated both in FP5and FP6 projects, there is still a lack of essential informationin many areas on how environmental factors interfere withhuman health and well-being. We do not know to any greatextent how multiple environmental exposures occurring dur-ing the whole human lifecycle from the moment of concep-tion to aging and senescence can result in adverse healtheffects. Due to the complexity of the issues involved, only anintegrated approach focusing on multiple stressors, path-ways and effects, using scientific advances and tools such asecotoxicological tools and modelling, can at medium or longterm give us more definitive ideas as to the extent of envi-ronmental factors on the burden of disease at individual orpopulation level.

Justification and rationaleThe draft work programme 2007 will continue implementingthe E&H Action Plan through research. The ‘impact pathway’approach adopted in FP6 will be further strengthened. Theefforts to be supported in the first two years of the pro-gramme will build on work undertaken in previous pro-grammes. In FP7, special attention will be given to combinedeffects of stressors and the role of confounding factors. Thefocus will be on vulnerable groups and emerging issues willnot be neglected. The aim will be to generate new evidenceand at the same time develop/enhance models that can helpus in better understanding and foresee health and environ-ment interactions.

To continue supporting informed policy making, research willfocus on new and/or improved tools for prevention strategieswith emphasis on air pollution and epidemiological research,with applications in biomonitoring. The ultimate aim will be to

aid policy makers in identifying prevention strategies througha better understanding of the full chain of cause effect rela-tionships: sources – emissions – concentrations – exposuresand health effects. Research actions such as high-resolutionGIS or validation of biomarkers for epidemiological researchor biomonitoring, developed through new technologies basedon ‘omics’ methodologies, will aid the understanding of theprecise relationships between environment and disease.Ultimately, this information may allow us to identify ways toreduce or prevent disease by pinpointing biochemical andmolecular functions that have been perturbed by environ-mental stressors.

Emerging issues to be tackled include health impacts of glob-al change and certain man-made chemicals, keeping in mindrealistic exposure scenarios. On the effect side, focus will beon research dealing with health impacts that continue to beprevalent and may be increasing, such as respiratory andother effects of air pollution, to be tackled by establishing alarge European cohort study, declining reproductive health,the reasons for which remain uncertain, and neurodevelop-mental effects.

Short-term goalsThe work programme for the first call for proposals in FP7 willcontinue contributing to the goals of the E&H Action Plan bytackling existing knowledge gaps via a series of small andlarge-scale multidisciplinary research actions. The efforts tobe supported will partly build on work undertaken in previousframework programmes. Priority will be given to issueswhich were largely neglected in FP6. These issues includethe long-term impacts of air pollution (whether outdoor orindoor) and chemicals on human health. Research support forbiomonitoring pilot activity – an important activity of the E&HAction Plan – will be provided. Support for new and emergingissues and approaches have not been forgotten, such as theuse of GIS approaches to support environmental epidemiolo-gy and to investigate the health impacts of drought anddesertification. Specific efforts have been made to introduceinternational co-operation more strongly in the environmentand health area and to link to international initiatives such asGEO in which environment and health is also a well-identifiedpriority. The ‘impact pathway’ approach adopted in FP6 willtherefore be further strengthened.

Medium-term goalsLater calls for proposals will continue supporting environmentand health research by focusing on environmental stressorsof greatest importance for European populations. Particularfocus will be on vulnerable population groups such as chil-dren and ageing population. The approach taken is practical,combining projects on specific targeted issues such as healthimpacts of electromagnetic fields with those which will takea more integrated approach in support of policy making suchas integrated health risk assessment of exposure to multipleurban risk factors such as noise, air pollution, and chemicals,

Research News: MENDOSSeventh Framework Programme

Perspectives for environment and health research activity in theSeventh Framework Programme (FP7)



taking into account socio-economic factors. The generalaim will be to generate new evidence and at the same timedevelop/enhance risk and impact assessment models andtools that can help us in better understanding and decipher-ing health and environment interactions. The ultimate aim willbe to aid policy makers in identifying prevention strategiesthrough a better understanding of the full chain of cause-effect relationships: sources – emissions – concentrations –exposures and health effects – economic valuation. Short andmedium term priorities for research are presented inAnnex 2.

Cross-thematic approaches and linksIn FP6, environment and health research was implementedthrough three thematic areas: ‘Health’, ‘Food Quality &Safety’, and ‘Environment’ research.

In FP7, environment and health research has been identifiedas an own activity under Theme 6 ‘Environment (includingClimate Change)‘. The aim is to focus on multidisciplinaryresearch activities on interactions of environmental risk fac-tors and human health, taking into account exposures via dif-ferent exposure routes.

In Theme 1 ‘Health’, focused research on major diseases,such as cancer and cardiovascular diseases, including chron-ic diseases and public health research on disease prevention,will complement the E&H research activity. In addition,research on food, nutrition and diet-related diseases and dis-orders will be the subject of Theme 2 ‘Food, Agriculture andBiotechnology’. These areas are seen more complementaryto the approach taken in the environment and health area. Insome specific cases, possibilities for coordinated callsbetween the E&H activity and the other two themes could beenvisaged.

International collaboration will be enhanced via specific topicsespecially relevant to third countries. The research efforts willalso support the goals of specific international initiatives suchas GEO or other international activities, such as sustainabledevelopment or water and health initiatives. Efforts will bemade to enhance synergies between the EC Environment

and Health activity and similar efforts elsewhere, e.g., in theUSA, possibly via co-ordinated calls.

Work programme 2007To build up the work programme for 2007, the environmentand health research activity in FP7 is divided in three areas:

1. Health effects of exposure to environmental stressorsIndicative topics for first call:• Indoor air pollution in Europe: an emerging environmental

health issue• Environmental factors and their impact on reproduction

and development

2. Integrated approaches for environment and health riskassessmentIndicative topics for first call:• European network on human biomonitoring• European cohort on air pollution• Drought and desertification-related public health and

socio-economic impacts

3. Delivery of methods and decisions to support tools forrisk analysis and policy developmentIndicative topics for first call:• Geographical information systems in support for environ-

ment and health research• ERA-NET for environment and health

ConclusionTaken together, the multidisciplinary research efforts to besupported in FP7, linking environmental issues to health,should contribute in a significant manner to the main aim ofthe E&H Action Plan, namely understanding the linksbetween environmental stressors and health impacts, takinginto account individual susceptibilities and focusing on certainpriority health outcomes. Other key driving forces behind theadopted research strategy are the Earth Observation initia-tive, and the Sustainable Development Strategy with itsfocus on health impacts of environmental chemicals.Thematic Strategies on the Urban Environment and AirPollution as well as the Programme of Community Action inthe Field of Public Health are also important initiatives identi-fying future research needs.

The first calls are likely to be launched at the end of 2006 orthe beginning of 2007.


Contact:

Dr Tuomo Karjalainen/Dr Kirsi HaavistoEuropean Commission, DG ResearchDirectorate I (Environment)B-1049 Brussels, Belgium

[email protected]@ec.europa.eu

Note: The indicative topics and priorities given in this paperare subject to change.



Weybridge +10 workshopEuropean workshop on the impact of endocrine disrupters

The European Union (EU) has embarked on massive research efforts on endocrinedisrupters and to date has launched scientific projects worth more than 140 millionEuros. A lot of this research has now been completed, and new findings concern-ing the effects of chemicals have emerged. Progress has been made in pinpoint-ing human life stages particularly vulnerable to endocrine disruption, and new data

about male reproductive health and wildlife effects have come to light.

To review the implications of these new findings for EU policy making, an interna-tional workshop was held in Helsinki (November 8-10, 2006), ten years on from the1996 European Workshop on the Impact of Endocrine Disrupters on Human Healthand Wildlife (so-called Weybridge meeting). International experts and policy mak-ers from EU member states assessed the new research findings, identified knowl-edge gaps, defined future research priorities and considered the implications forpolicy making and chemicals regulation. The Helsinki workshop was held underFinland’s presidency of the EU and organised by the Academy of Finland, withcooperation from the European Commission's Research Directorate-General andthe European Environment Agency.

Results of the 2006 Helsinki workshop will be published as a consensus document.For more information see www.aka.fi/euseminars

Copenhagen workshop on endocrine disruptersConsumer products and endocrine disrupters: possible effects onhuman populations

May 28 – May 31, 2007, Copenhagen, Denmark

The fourth Copenhagen workshop on endocrine disrupters to be held atRigshospitalet is intended to facilitate an exchange of information and views bothwithin the scientific community and with experts engaged in regulation and policy-making. The workshop will focus on possible effects of exposures to endocrine dis-rupters present in our everyday life e.g. in our food, cosmetic and our homes. Therole of mixed exposures and links between effects in laboratory animals and obser-vations in wildlife and humans will also be addressed. Effects both on reproductionand non-reproductive organs will be discussed. The meeting is supported by theDanish Ministry of the Environment. More information is available atwww.reproduction.dk

Endocrine disrupting contaminantsdiet interaction database (EDID)

Diet is a major source of exposure to contaminants such as endocrine disrupters.It is also a major modifier of the organism’s susceptibility to xenobiotics, throughthe intake (adequate, unbalanced or deficient) of nutrients and ‘natural’ bioactivesubstances. Limited knowledge is available on the interactions among xenobioticsand substances naturally present in food commodities, even though more infor-mation would be highly relevant to the more general issues of food safety andprevention.

The Italian Istituto Superiore di Sanità (ISS) website on endocrine disrupters hasbeen updated with a section dedicated to a brand new database named EDID:Endocrine Disrupting Contaminants Diet Interaction Database. EDID is a databaseof studies present in the international literature, either on experimental systems,on animal populations or humans, which aims to be easy to consult, periodicallyupdated and to stimulate further research in this field. EDID has been created aspart of the ISS special project on endocrine disrupters. The database may beaccessed at www.iss.it/inte/edid/cont.php?id=110&lang=2&tipo=17

Participating projectsCOMPRENDOCoordinator: Dr Ulrike Schulte-Oehlmannwww.comprendo-project.org

EDENCoordinator: Dr Andreas Kortenkampwww.edenresearch.info

EURISKEDCoordinator: Prof. Wolfgang Wuttke

www.eurisked.orgFIRECoordinator: Prof. Antoon Opperhuizenwww.rivm.nl/fire

Associated projectsACEBONETOXEASYRINGEDERA

See www.credocluster.info

CREDO coordinationFor further information and to be keptinformed of developments within CREDOplease contact:

Dr Andreas [email protected]/fax: +44 20 7753 5908

Dr Ragnor [email protected]/fax: +44 20 7753 5811

Centre for ToxicologyThe School of Pharmacy,University of London29-39 Brunswick Square, London WC1N 1AXUnited Kingdom

www.credocluster.info

Contact at the European Commission:

Dr Tuomo [email protected]

Dr Kirsi [email protected]

© Dr Andreas Kortenkamp, 2006

DisclaimerWhile every effort has been made to providecorrect information, we assume noresponsibility for the accuracy of theinformation. Although this describes EU-fundedprojects, neither the European Union nor theEuropean Commission shall be held responsiblefor the content, nor the views expressed here.

The CREDO cluster is fundedby the European Commission’sFifth Framework Programme forresearch, technologicaldevelopment and demonstrationactivities in the EuropeanCommunity.

A joint project funded by twothematic programmes:Quality of Life and Managementof Living Resources Programme,and Energy, Environmentand Sustainable Development

ENDOMETGENDISRUPTMENDOSSENSPESTI

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